UXT potentiates angiogenesis by attenuating Notch signaling.
Significantly, UXT attenuates the expression of the Notch-responsive genes in vitro and in vivo.Mechanistically, UXT binds to the promoters of the Notch signaling target genes and specifically interacts with the transactivation region domain of the Notch intracellular domain (NICD), impairing the interaction between NICD and the transcription factor RBP-Jκ endogenously.This prevents RBP-Jκ/CSL from activation and thus inhibits the consequent gene inductions.
Affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
- Neovascularization, Physiologic/genetics/physiology*
- Receptors, Notch/genetics/metabolism*
- Zebrafish Proteins/genetics/metabolism*
- Cell Division/genetics/physiology
- Cell Movement/genetics/physiology
- Human Umbilical Vein Endothelial Cells
- Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics/metabolism
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DEV112532F3: UXT is indispensable for zebrafish ISV spouting. (A-F) Confocal imaging of the UXT-deficient Tg(kdrl:EGFP)s843 embryos at 30 hpf. (A,C,E) Bright-field images merged with confocal images. (B,D,F) Higher magnification images of the confocal-imaged area in the red boxed regions in A,C and E. (A) General morphology and trunk vascular phenotype and (B) the ISV sprouts in a Tg(kdrl:EGFP)s843 embryo injected with 4 ng of control morpholino (Ctrl MO). (C) General morphology and trunk vascular phenotype, and (D) the ISV sprouts in a Tg(kdrl:EGFP)s843 embryo injected with 4 ng of UXT morpholino (UXT MO). (E) General morphology and trunk vascular phenotype, and (F) the ISV sprouts in a Tg(kdrl:EGFP)s843 embryo injected with 6 ng of UXT MO. The ISVs are marked by the vertical white line. The yellow and red bars indicate the lumen of the dorsal aorta (DA) and posterior cardinal vein (PCV), respectively. All of the embryos were confocal imaged at 30 hpf and are shown in lateral views with rostral left and dorsal up. Scale bar:100 µm. (G-J) Confocal imaging of the Tg(kdrl:EGFP)s843 morphants rescued by UXT mRNA. (H,J) The confocal imaged area of the red boxed regions in G and I. (G) The trunk vascular phenotype and (H) the ISV sprouts in a Tg(kdrl:EGFP)s843 embryo injected with 4 ng of UXT morpholino plus 150 pg of wild-type UXT mRNA (4 ng UXT MO+150 pg UXT mRNA). (I) The trunk vascular phenotype and (J) the ISV sprouts in a Tg(kdrl:EGFP)s843 embryo injected with 4 ng of UXT MO plus mutant UXT mRNA (4 ng UXT MO+150 pg UXT-2M mRNA). All of the embryos were confocal imaged at 30 hpf. Scale bar: 100 µm. (K) Statistical analysis of the ISV length and the lumen diameter of the DA or PCV from embryos injected with Ctrl MO, UXT MO, UXT MO+UXT mRNA or UXT MO+UXT-2M mRNA. For one experiment, six embryos of each treatment were analyzed at 30 hpf. Data show the mean±s.e.m. (at least three independent experiments); ***P<0.001 versus the corresponding control. (L) The whole-mount in situ hybridization of UXT-deficient embryos at 30 hpf. Riboprobes are against the arterial markers efn2a, notch1, notch3, tbx20, hey2 and the venous markers aplnra, flt4 and efnb4. The yellow arrowheads denote the DA; the yellow asterisks indicate the PCV. (M-R) Confocal imaging of the donor Tg(kdrl:EGFP)s843 cells in non-transgenic embryos at 30 hpf. (M) Wild-type cells contribute to all trunk vessels, including the DA (asterisk), PCV (red arrowhead), basal cells (red bracket), connector cells (white bracket) and DLAV (yellow arrowhead). (P) Deficient for UXT, the endothelial cells of Tg(kdrl:EGFP)s843 can only contribute to theDA, basal cells and connector cells. (N,Q) The transplanted donor cells were stained with Rhodamine-561 dextran. (O,R) Merged images of EGFP and Rhodamine-561 dextran. Scale bar: 75 µm. (S,T) Quantification of wild-type (S) and UXT-deficient (T) donor Tg(kdrl:EGFP)s843 cells in non-transgenic embryos. n, the number of successfully transplanted embryos.
To further dissect the function of UXT in angiogenesis, we analyzed the ISV development of Tg(kdrl:EGFP)s843 transgenic zebrafish using confocal microscopy. As expected, ISVs of control embryos extended to the dorsal-most aspect of the neural tube at 30 hpf, and T-shape branches formed by dorsal longitudinal anastomotic vessels (DLAV) were normal (Fig. 3A,B). Remarkably, ISVs of the embryos that were injected with 4 ng of UXT MO sprouted to the horizontal myoseptum and did not reach the dorsal roof to form DLAVs (Fig. 3C,D). Furthermore, the embryos that were injected with 6 ng of UXT MO displayed severe defects, such as a shorter trunk and a more downward curly tail (Fig. 3E). Notably, the ISVs of UXT-deficient embryos projected up at the midline of the trunk, and almost no ISVs were generated at posterior part of the trunk (Fig. 3E,F).Fig. 3.